The present invention relates to methods for separating or isolating a biological target material from other substances in a medium to produce an isolated material of sufficient purity for further processing or analysis. The present invention particularly relates to methods for separating or isolating biological target materials using magnetically responsive particles capable reversibly binding the material. The present invention more specifically relates to methods for separating or isolating biological target materials using at least one magnetically responsive particle comprising silica or a silica derivative such as silica gel which reversibly binds the biological target material thereof.
Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, and sequencing involve the processing or analysis of biological materials. These techniques generally require that such materials be substantially free of contaminants capable of interfering with such processing or analysis procedures. Such contaminants generally include substances that block or inhibit chemical reactions, (e.g. nucleic acid or protein hybridizations, enzymatically catalyzed reactions, and other types of reactions, used in molecular biological techniques), substances that catalyze the degradation or depolymerization of a nucleic acid or other biological material of interest, or substances that provide xe2x80x9cbackgroundxe2x80x9d indicative of the presence in a sample of a quantity of a biological target material of interest when the nucleic acid is not, in fact present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a nucleic acid material of interest is isolated, macromolecular substances such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors or oligonucleotides. Contaminants can also be introduced into a target biological material from chemicals or other materials used to isolate the material from other substances. Common contaminants of this last type include trace metals, dyes, and organic solvents.
Obtaining DNA or RNA sufficiently free of contaminants for molecular biological applications is complicated by the complex systems in which the DNA or RNA is typically found. These systems, e.g., cells from tissues, cells from body fluids such as blood, lymph, milk, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out, typically include significant quantities of contaminants from which the DNA or RNA of interest must be isolated before being used in a molecular biological procedure.
Conventional protocols for obtaining DNA or RNA from cells are described in the literature. See, e.g. Chapter 2 (DNA) and Chapter 4 (RNA) of F. Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley-Interscience, New York (1993). Conventional DNA isolation protocols generally entail suspending the cells in a solution and using enzymes and/or chemicals, gently to lyse the cells, thereby releasing the DNA contained within the cells into the resulting lysate solution. For isolation of RNA, the conventional lysis and solubilization procedures include measures for inhibition of ribonucleases and contaminants to be separated from the RNA including DNA.
Many conventional protocols in use today also generally entail use of phenol or an organic solvent mixture containing phenol and chloroform to extract additional cellular material such as proteins and lipids from a conventional lysate solution produced as described above. The phenol/chloroform extraction step is generally followed by precipitation of the nucleic acid material remaining in the extracted aqueous phase by adding ethanol to that aqueous phase. The precipitate is typically removed from the solution by centrifugation, and the resulting pellet of precipitate is allowed to dry before being resuspended in water or a buffer solution for further processing or analysis.
Conventional nucleic acid isolation procedures have significant drawbacks. Among these drawbacks are the time required for the multiple processing steps necessary in the extractions and the dangers of using phenol or chloroform. Phenol causes severe burns on contact. Chloroform is highly volatile, toxic and flammable. Those characteristics require that phenol be handled and phenol/chloroform extractions be carried out in a fume hood.
Another undesirable characteristic of phenol/chloroform extractions is that the oxidation products of phenol can damage nucleic acids. Only freshly redistilled phenol can be used effectively, and nucleic acids cannot be left in the presence of phenol. Generally also, multi-step procedures are required to isolate RNA after phenol/chloroform extraction. Ethanol (or isopropanol) precipitation must be employed to precipitate the DNA from a phenol/chloroform-extracted aqueous solution of DNA and remove residual phenol and chloroform from the DNA. Further, ethanol (or isopropanol) precipitation is required to remove some nucleoside triphosphate and short (i.e., less than about 30 bases or base pairs) single or double-stranded oligonucleotide contaminants from the DNA. Moreover, under the best circumstances such methods produce relatively low yields of isolated nucleic acid material and/or isolated nucleic acid material contaminated with impurities.
There is a need recognized in the art for methods, that are simpler, safer, or more effective than the traditional phenol/chloroform extraction/ethanol precipitation methods to isolate DNA and/or RNA sufficiently for manipulation using molecular biological procedures.
Fractionation of DNA recovered from cells according to size is required for many molecular biological procedures. Such fractionation is typically accomplished by agarose or polyacrylamide gel electrophoresis. For analysis or treatment by a molecular biological procedure after fractionation, the DNA in the fraction(s) of interest must be separated from contaminants, such as agarose, other polysaccharides, polyacrylamide, acrylamide, or acrylic acid, in the gel used in such electrophoresis. Thus, there is also a need in the art for methods to accomplish such separations.
Methods for amplifying nucleic acids or segments thereof, such as the well known polymerase chain reaction (PCR) process (see, e.g., U.S. Pat. No. 4,683,202), yield solutions of complex mixtures of enzymes, nucleoside triphosphates, oligonucleotides, and other nucleic acids. Typically, the methods are carried out to obtain an highly increased quantity of a single nucleic acid segment (xe2x80x9ctarget segmentxe2x80x9d). Often it is necessary to separate this target segment from other components in the solution after the amplification process has been carried out. Thus there is a further need in the art for simple methods to accomplish these separations.
Silica materials, including glass particles, such as glass powder, silica particles, and glass microfibers prepared by grinding glass fiber filter papers, and including diatomaceous earth, have been employed in combination with aqueous solutions of chaotropic salts to separate DNA from other substances and render the DNA suitable for use in molecular biological procedures. See U.S. Pat. No. 5,075,430 and references cited therein, including Marko et al., Anal. Biochem. 121, 382-387 (1982) and Vogelstein et al., Proc. Natl. Acad. Sci. (USA) 76, 615-619 (1979). See also Boom et al., J. Clin. Microbiol. 28, 495-503 (1990). With reference to intact glass fiber filters used in combination with aqueous solutions of a chaotropic agent to separate DNA from other substances, see Chen and Thomas, Anal. Biochem. 101, 339-341 (1980). Vogelstein et al., supra, suggest that silica gel is not suitable for use in DNA separations. With regard to separation of RNA using silica materials and chaotropic agents, see Gillespie et al., U.S. Pat. No. 5,155,018.
Glass particles, silica particles, silica gel, and mixtures of the above have been configured in various different forms to produce matrices capable of reversibly binding nucleic acid materials when placed in contact with a medium containing such materials in the presence of chaotropic agents. Such matrices are designed to remain bound to the nucleic acid material while the matrix is exposed to an external force such as centrifugation or vacuum filtration to separate the matrix and nucleic acid material bound thereto from the remaining media components. The nucleic acid material is then eluted from the matrix by exposing the matrix to an elution solution, such as water or an elution buffer. Numerous commercial sources offer silica-based matrices designed for use in centrifugation and/or filtration isolation systems. See, e.g. Wizard(trademark) DNA purification systems line of products from Promega Corporation (Madison, Wis., U.S.A.); or the QiaPrep(trademark) line of DNA isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.)
Magnetically responsive particles (hereinafter, xe2x80x9cmagnetic particlesxe2x80x9d) have conventionally been used to isolate and purify polypeptide molecules such as proteins or antibodies. In recent years, however, magnetic particles and methods for using magnetic particles have been developed for the isolation of nucleic acid materials. Several different types of magnetic particles designed for use in nucleic acid isolation are described in the literature, and many of those types of particles are available from commercial sources. Such magnetic particles generally fall into either of two categories, those designed to reversibly bind nucleic acid materials directly, and those designed to do so through at least one intermediary substance. The intermediary substance is referred to herein as a xe2x80x9clabel.xe2x80x9d
The magnetic particles designed to bind nucleic acid materials indirectly are generally used to isolate a specific nucleic acid material, such as mRNA, according to the following basic isolation procedure. First, a medium containing a nucleic acid material is placed in contact with a label capable of binding to the nucleic acid material of interest. For example, one such commonly employed label, biotinylated oligonucleotide deoxythymidine (oligo-dT), forms hydrogen bonds with the poly-adenosine tails of mRNA molecules in a medium. Each label so employed is designed to bind with a magnetically responsive particle, when placed into contact with the particle under the proper binding conditions. For example, the biotin end of a biotinylated oligo-dT/mRNA complex is capable of binding to streptavidin moieties on the surface of a streptavidin coated magnetically responsive particle. Several different commercial sources are available for streptavidin magnetic particles and reagents designed to be used in mRNA isolation using biotinylated oligo-dT as described above. See, e.g. PolyATtract(copyright) Series 9600(trademark) mRNA Isolation System from Promega Corporation; or the ProActive(trademark) line of streptavidin coated microsphere particles from Bangs Laboratories (Carmel, Ind., U.S.A.). Magnetic particles and label systems have also been developed which are capable of indirectly binding and isolating other types of nucleic acids, such as double-stranded and single-stranded PCR templates. See, e.g. BioMag(trademark) superparamagnetic particles from Advanced Magnetics, Inc. (Cambridge, Mass., U.S.A.)
Indirect binding magnetic separation systems for nucleic acid isolation or separation all require at least three components, i.e. magnetic particles, a label, and a medium containing the nucleic acid material of interest. The label/nucleic acid binding reaction and label/particle binding reaction often require different solution and/or temperature reaction conditions from one another. Each additional component or solution used in the nucleic acid isolation procedure adds to the risk of contamination of the isolated end product by nucleases, metals, and other deleterious substances.
A few types of magnetic particles have also been developed for use in the direct binding and isolation of biological materials, particularly nucleic acid. One such particle type is a magnetically responsive glass bead, preferably of a controlled pore size. See, e.g. Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park, N.J., U.S.A.); or porous magnetic glass particles described in U.S. Pat. Nos. 4,395,271; 4,233,169; or 4,297,337. Nucleic acid material tends to bind so tightly to glass, however, that it can be difficult to remove once bound thereto. Therefore, elution efficiencies from magnetic glass particles tend to be low compared to elution efficiencies from particles containing lower amounts of a nucleic acid binding material such as silica.
A second type of magnetically responsive particles designed for use in direct binding and isolation of biological materials, particularly nucleic acid, are particles comprised of agarose embedded with smaller ferromagnetic particles and coated with glass. See, e.g. U.S. Pat. No. 5,395,498. A third type of magnetically responsive particle, a particle capable of directly bind enzymes, proteins, hormones, or antibodies, is produced by incorporating magnetic materials into the matrix of polymeric silicon dioxide compounds. See, e.g. German Patent No. DE 43 07 262 A1. The latter two types of magnetic particles, the agarose particle and the polymeric silicon dioxide matrix, tend to leach iron into a medium under the conditions required to bind biological materials directly to each such magnetic particle. It is also difficult to produce such particles with a sufficiently uniform and concentrated magnetic capacity to ensure rapid and efficient isolation of nucleic acid materials bound thereto.
What is needed is a method for isolating biological entities, particularly nucleic acids, using a magnetically responsive particle capable of rapidly and efficiently directly isolating such entities sufficiently free of contaminants to be used in molecular biology procedures.
Briefly, in one aspect, the present invention comprises a method of isolating a biological target material from other materials in a medium by:
providing a medium including the biological target material;
providing silica magnetic particles;
forming a complex of the silica magnetic particles and the biological target material by combining the silica magnetic particles and the medium;
removing the complex from the medium by application of an external magnetic field and
separating the biological target material from the complex by eluting the biological target material whereby the isolated biological target material is obtained.
In a further aspect, the present invention is a method of isolating a biological target material of interest from other materials in a medium using silica magnetic particles capable of reversibly binding at least 2 micrograms of biological target material per milligram of silica magnetic particles, and of releasing at least 60% of the biological target material bound thereto. In preferred practices of the present method, at least about 4 micrograms of biological target material per milligram of silica magnetic particle is bound thereto and at least about 75% of the biological target material adhered to the silica magnetic particles is subsequently eluted. The biological target material isolated according to the method of this invention is preferably nucleic acid.
A preferred practice of the method of the present invention comprises the following steps. First, a mixture is formed comprising the medium and the silica magnetic particles. Second, the biological target material is adhered to the silica magnetic particles in the mixture. Third, the silica magnetic particles are removed from the mixture using an external force, most preferably using a magnetic force, and Fourth, at least 60% of the biological target material adhered to the silica magnetic particle is eluted by contacting the particle with an elution solution.
In another aspect, the present invention is a method of isolating plasmid DNA from other materials in a medium using a preferred form of silica magnetic particle, i.e., siliceous-oxide coated magnetic particle, wherein the preferred particles are capable of binding at least 2 micrograms of the plasmid DNA material per milligram of particle, and of releasing at least 60% of the plasmid DNA material bound thereto. A preferred practice of the methods of this aspect of the invention comprise the following steps. First, a mixture is formed comprising the medium including plasmid DNA, the siliceous-oxide coated magnetic particle, and a chaotropic salt. Second, the plasmid DNA is adhered to the siliceous-oxide coated magnetic particle in the mixture. Third, the siliceous-oxide coated magnetic particle is removed from the mixture using an external force, most preferably using a magnetic field. Fourth, at least 60% of the plasmid DNA adhered to the siliceous-oxide coated magnetic particle is eluted by contacting the particle with an elution solution.
In a further aspect, the present invention is a kit for isolating a biological target material from a medium containing the same, the kit comprising an aliquot of siliceous-oxide coated magnetic particles suspended in an aqueous solution in a first container, wherein the particles have the capacity to reversibly bind at least 2 micrograms of the biological target material per milligram of particle. Optionally, the kit may include other components needed to isolate a biological target material from a medium containing the same according to the methods of the present invention.
As used herein, the term xe2x80x9cmagnetic particlesxe2x80x9d refers to materials which have no magnetic field but which form a magnetic dipole when exposed to a magnetic field, i.e., materials capable of being magnetized in the presence of a magnetic field but which are not themselves magnetic in the absence of such a field. The term xe2x80x9cmagneticxe2x80x9d as used in this context includes materials which are paramagnetic or superparamagnetic materials. The term xe2x80x9cmagneticxe2x80x9d, as used herein, also encompasses temporarily magnetic materials, such as ferromagnetic or ferrimagnetic materials with low Curie temperatures, provided that such temporarily magnetic materials are paramagnetic in the temperature range at which silica magnetic particles containing such materials are used according to the present methods to isolate biological materials.
The term xe2x80x9csilica magnetic particlexe2x80x9d refers to a magnetic particle comprised of silica in the form of silica gel, siliceous oxide, solid silica such as glass or diatamaceous earth, or a mixture of two or more of the above. The term xe2x80x9csilica gelxe2x80x9d as used herein refers to chromatography grade silica gel, a substance which is commercially available from a number of different sources. Silica gel is most commonly prepared by acidifying a solution containing silicate, e.g. sodium silicate, to a pH of less than 10 or 11 and then allowing the acidified solution to gel. See, e.g. silica preparation discussion in Kurt-Othmer Encyclopedia of Chemical Technology, Vol. 6, 4th ed., Mary Howe-Grant, ed., John Wiley and Sons, pub., 1993, pp. 773-775. The term xe2x80x9csilica magnetic particlexe2x80x9d as used herein preferably refers to particles with the characteristics described above having the capacity to bind at least 2 micrograms of biological target material per milligram of silica magnetic particles and, independently, the capacity to release at least 60% of the biological target material bound thereto in the elution step of the present method. The silica magnetic particles used in the present invention preferably further comprise ferromagnetic material incorporated into a silica gel matrix. The elution step in the isolation methods of this invention are preferably accomplished without substantial contamination of the nucleic acid material by metal or metal compounds (e.g., iron or iron compounds) or other objectionable species originating from the silica magnetic particles.
The term xe2x80x9cglass particlesxe2x80x9d as used herein means particles of crystalline silicas (e.g., xcex1-quartz, vitreous silica), even though crystalline silicas are not formally xe2x80x9cglassesxe2x80x9d because they are not amorphous, or particles of glass made primarily of silica.
The term xe2x80x9csiliceous-oxide coated magnetic particlexe2x80x9d or xe2x80x9cSOCM particlexe2x80x9d is used herein to refer to the most preferred form of silica magnetic particle used in the methods and kits of the present invention. The SOCM particle is comprised of siliceous oxide coating a core of at least one particle of superparamagnetic or paramagnetic material. The SOCM particle used in the present method and kits also has an adsorptive surface of hydrous siliceous oxide, a surface characterized by having silanol groups thereon. Target nucleic acid material, such as DNA or RNA, adhere to the adsorptive surface of the particle while other material, particularly deleterious contaminants such as exonucleases, do not adhere to or co-elute from the particle with the nucleic acid materials. The physical characteristics of the SOCM particle and methods for producing such particles are disclosed in concurrently filed U.S. patent application Ser. No. 08/786,600, entitled xe2x80x9cSilica Adsorbent on Magnetic Substrate,xe2x80x9d the disclosure of which is incorporated by reference herein.
The present invention provides convenient and efficient means for isolating biological target material of interest from a variety of different media. A preferred aspect of the present method described briefly above, wherein magnetic force is used to remove the particles from the media, offers significant advantages over conventional isolation methods wherein a biological target material is reversibly bound to other silica material. Specifically, the magnetic removal step of the method substitutes for vacuum filtration or centrifugation steps required in conventional silica binding and elution isolation methods. It is, therefore, particularly amenable to being automated. Small laboratories or individual researchers frequently must purchase specialized and expensive equipment to carry out such methods, such as a vacuum manifold and vacuum for use in vacuum filtration or a microcentrifuge for centrifugation methods. Contrastingly, magnetic separation of the present invention merely requires a concentrated magnetic field such as is generated from a strong and readily available magnet. Inexpensive apparatuses specifically adapted for use in molecular biology research context are also commercially available, such as the MagneSphere(copyright) Technology Magnetic Separation Stand or the PolyATract(copyright) Series 9600(trademark) Multi-Magnet (both available from Promega Corporation, Madison, Wis., USA).
The biological target material isolated using the isolation method of the present invention is sufficiently free of contaminating material for additional processing or analysis using standard molecular biology techniques. Applications of the present methods to isolate various different biological target materials from a variety of different media will become apparent from the detailed description of the invention below.